In addition to conventional antibodies, each of which has two heavy and two light chains, camelids (old-world and new-world camels) also produce antibodies consisting exclusively of heavy chains. These so-called heavy-chain antibodies are homodimers of two identical heavy chains having a single variable domain for interacting with an antigen, known as VHH (variable domain of the heavy chain of heavy chain antibodies).
Thus camelids have IgG subclasses (IgG2+3) which have a VHH fragment instead of a Fab fragment. This VHH fragment offers various advantages in terms of antigen-binding behavior, but especially has the particular advantage of being able to be produced by a simple recombinant process in various expression organisms such as bacteria and yeasts. Likewise, antigen-binding VHH fragments can be produced synthetically. These recombinant VHH fragments are also known under the trade name of Nanobodies®.
The VHH domain has three complementarity-determining regions (CDRs) and four framework regions (FR). The VHH domain of a heavy chain antibody of this type forms a very small polypeptide unit characterized by a high antigen binding capacity, and therefore these fragments are of great interest for therapeutic and diagnostic purposes.
However, the VHH fragments cannot be used as complete antibodies, since the FC fragment is lacking. In therapeutic application this results in a shortened half life, and many laboratories do not have the appropriate secondary reagents for use in immune test systems such as ELISA.
In some instances the missing FC fragment is an advantage in use, but there are also applications in which coupling to an FC fragment would be advantageous. A human FC fragment is needed especially in immunotherapeutic applications. Problems often arise in the preclinical testing of antibody products, since the test product has a human FC fragment, but the preclinical studies were conducted in nonhuman organisms. The immune system responds very differently to FC fragments from other species. Therefore it would be advantageous if a new therapeutic agent in preclinical testing would be equipped with the respective FC fragment of the test species, but otherwise did not differ in any way from the product to be used in humans.
Monoclonal antibodies, most frequently monoclonal mouse antibodies, are often used in immune test systems. The development and production of monoclonal mouse antibodies is associated with great effort and expense. The preparation of recombinant and thus monoclonal VHH fragments can be done much more advantageously and efficiently. However, it would be advantageous for the immune test systems if an FC fragment of an antibody frequently used in such test systems were available in order to continue to work with the same protocols, reagents and methods.
To recreate complete immunoglobulins from the recombinantly produced VHH fragments, up to now the whole antibody was produced by recombinant means. In this process the Fab fragment is replaced by the VHH fragment. This method is primarily used for therapeutic purposes, and correspondingly, a human Fc fragment is used, as a rule. The production of such recombinant, chimeric antibodies can only be done in mammalian cells, and therefore the advantage of cost-advantageous, large-scale production of VHH fragments in bacteria or yeasts is lost.
VHH fragments are very small and on average have a molecular weight of about 12-15 kDa. VHH fragments as a medication have a very short plasma half-life unless modifications are made to prevent rapid renal clearance. For this purpose either serum protein linkers (e. g., HSA) may be used, or the entire construct can be enlarged by preparing bivalent or multivalent constructs. Another possibility is PEGylation of the protein.
VHH fragments are also used for research. Here they offer the advantage of being very small. Precisely for this reason, specially labeled VHH fragments are a valuable tool, particularly in live cell imaging. However, they are not usable for many immunologic detection methods, since either corresponding secondary reagents do not exist at all, or they are not available for the specific purpose.